Fig. 3

Bleomycin impairs DSB repair efficiency by HR. A HEK293T-spCas9 cells were co-transfected with sgRNA and dsDNA/dsODN, followed by treatment of bleomycin for 48 h, and genomic DNA was extracted for RT-PCR analysis. The HR-suppressive effects of bleomycin were shown in a dose-dependent manner. B Measurement of HR and NHEJ activities by using the GFP-based HR/NHEJ reporter assay. HEK293T-spCas9 cells were transfected with I-SceI-linearized plasmids and incubated with bleomycin at different concentrations for 48 h. A DsRed-expressing plasmid was co-transfected as transfection control. The ratio of GFP-positive cells to DsRed-positive cells was measured by flow cytometry. C A549 cells were treated with the indicated concentrations of bleomycin for 24 h, the expression levels of important HR (left) and NHEJ (right) pathway-associated factors were analyzed by Western blotting. Significance markers: *p < 0.05; **p < 0.01 compared to control; n = 3